Generation of cytotoxic tumor specific cell lines and uses thereof

ABSTRACT

An in-vitro method of activating T cells is disclosed. The method comprises incubating T cells with pathogenic cells in the presence of a multimeric peptide comprising at least two peptide monomers linked to one another, each of the at least two peptide monomers comprising at least 6 consecutive amino acids from the amino acid sequence as set forth in SEQ ID NO: 1, wherein the at least two peptide monomers are each no longer than 30 amino acids, wherein the multimeric peptide is capable of reducing binding of PLIF to human leukocytes under conditions which allow expansion of the T cells.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.14/765,570 filed on Aug. 4, 2015 which is a National Phase of PCT PatentApplication No. PCT/IL2014/050114 having International filing date ofFeb. 3, 2014, which claims the benefit of priority under 35 USC § 119(e)of U.S. Provisional Patent Application No. 61/760,215 filed on Feb. 4,2013. The contents of the above applications are all incorporated byreference as if fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 74378SequenceListing.txt, created on Jun. 18,2018, comprising 22,302 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates togeneration of cytotoxic tumor specific cell lines using PlacentalImmunoregulatory Ferritin (PLIF) peptides.

Clinical studies on tumor cell-based vaccines are based on the conceptthat autologous or allogeneic tumor cells express many tumor-associatedantigens (TAA). The MHC class I system, along with the endogenouspeptides presented on the cell surface are unique markers used byeffector CD8+ T cells to discriminate normal cells from diseased cells.MHC class I complexes are constitutively expressed by all nucleatedcells in the body. The MHC system includes Ag-processing machinery thatprocesses and presents peptides in the context of MHC molecules on tothe cell surface. Thus, cytotoxic T cells made against TAA complexes(TAA/MHC) that mediate anti tumor effects could serve as a novelmodality for cancer treatment.

In order to develop immunotherapy for cancer, it is of the utmostimportance to have representative target cell lines that presentrelevant levels of peptides from TAAs on HLA class I molecules. SinceHLA-A*0201 is the most common HLA class I molecule in humans, moststudies describing the generation of T cells against cancer cell TAAshave focused on HLA-A*0201-restricted peptides.

Several human studies in recent years have demonstrated that theinfusion of tumor-specific cytotoxic T cell lines and clones may have apositive clinical effect on diverse malignant diseases, such ascolorectal cancer, Hodgkin's lymphoma and nasopharyngeal carcinoma. Inmost studies documented, the amount of cytotoxic T cell lines requiredfor therapy range from 1×10⁷ to 1×10⁸ per infusion, and most treatmentregimens require several cycles of adoptive transfer.

Tumor-specific cytotoxic lymphocytes are usually expanded fromperipheral blood mononuclear cells (PBMC) taken from tumor-bearingpatients. These are expanded using antigen-presenting cells pulsed withirradiated tumor cells, tumor peptides, tumor lysates or fused tumorcells, resulting in the expansion of MHC class I-restricted cytotoxic Tcell lines over several weeks of culture. Tumor-specific cytotoxic Tcell lines can also be derived as a subpopulation of tumor-infiltratinglymphocytes by modifying the methodologies, including a purificationstep based on the selection of CD8 T cells.

In human clinical trials, infusion of tumor-specific T cells derivedfrom tumor-infiltrating lymphocytes or draining lymph nodes has shownlimited but encouraging clinical responses in specific settings.Unfortunately, the ability to expand tumor antigen-specific T cells exvivo from cancer patients is technically difficult due to numerousobstacles, including initiating cultures with low numbers oftumor-specific T cells and the physical inability to obtaintumor-infiltrating lymphocytes from patients with the most commonmalignancies.

Placenta Immunomodulatory Factor (PLIF) is a protein composed of 165amino acids. Of these, 117 match the ferritin heavy chain sequence,whereas the C-terminal 48 amino acids (C48) has a sequence which is notrelated to ferritin. It has been shown that the subcloned recombinantC48 peptide exhibits the bioactivity and therapeutic properties of PLIF[Moroz et al, J. Biol. Chem. 2002, 277, 12901-12905].

PLIF is expressed in the feto-maternal interface in both decidualmononuclear cells and syncytiotrophoblast cells. C48/PLIF binds tomacrophages and activated T cells, inducing high levels of IL-10, andacts as a regulatory cytokine. It governs the balance between Th1/Th2cytokines, which is essential for induction of tolerance duringpregnancy. A significantly high correlation was observed between lowlevels of serum PLIF and the different pathological pregnancyconditions: early pregnancy failures; pregnancies complicated withabortion; intrauterine growth restriction (IUGR); and women at risk fordeveloping pre-eclampsia.

It has been shown that PLIF is upregulated and expressed in malignantcells such as Hodgkin's and non-Hodgkin's lymphoma, acute lymphaticleukemia (ALL), human breast cancer tissues, and breast cancer celllines (T47D and MCF-7), but not in benign breast disease. Similar to theembryo, PLIF manipulates the cytokine network and immune response,enabling immune escape.

Experiments have been performed to restore T cell immunity and inducerejection of breast cancer by neutralizing C48/PLIF. Rabbit anti-C48polyclonal antibodies injected intraperitoneally (i.p.) into immunecompromised Nude mice engrafted with MCF-7 human breast cancer cellsresulted in growth arrest associated with human cell apoptosis andmassive intra-tumor lymphocytic infiltration. This was accompanied byactivation of INF-γ, thus affecting the cytokine network and leading tobreakdown of tolerance.

Synthetic PLIF dimeric peptides are disclosed in U.S. Patent ApplicationNo. 61/614,110 for the treatment of cancer.

Additional background art includes U.S. Pat. No. 4,882,270 whichdiscloses a method for detecting breast cancer, by using antibodiesagainst isoferritin placental protein.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an in-vitro method of activating T cells, the methodcomprising incubating T cells with pathogenic cells in the presence of amultimeric peptide comprising at least two peptide monomers linked toone another, each of the at least two peptide monomers comprising atleast 6 consecutive amino acids from the amino acid sequence as setforth in SEQ ID NO: 1, wherein the at least two peptide monomers areeach no longer than 30 amino acids, wherein the multimeric peptide iscapable of reducing binding of PLIF to human leukocytes under conditionswhich allow expansion of the T cells.

According to an aspect of some embodiments of the present inventionthere is provided an in vitro method of increasing the cytotoxicity of Tcells comprising incubating pathogenic cells which have an upregulatedamount of Placenta Immunomodulatory Factor (PLIF) as compared to healthycells with T cells in the presence of a multimeric peptide comprising atleast two peptide monomers linked to one another, each of the at leasttwo peptide monomers comprising at least 6 consecutive amino acids fromthe amino acid sequence as set forth in SEQ ID NO: 1, wherein the atleast two peptide monomers are each no longer than 30 amino acids,wherein the multimeric peptide is capable of reducing binding of PLIF tohuman leukocytes under conditions which allow for the generation ofactivated T cells that are cytotoxic to the pathogenic cells, therebyincreasing the cytotoxicity of the T cells.

According to an aspect of some embodiments of the present inventionthere is provided an in vitro method of generating a cytotoxic T cellline comprising:

(a) incubating pathogenic cells which have an upregulated amount ofPlacenta Immunomodulatory Factor (PLIF) as compared to healthy cellswith T cells in the presence of a multimeric peptide comprising at leasttwo peptide monomers linked to one another, each of the at least twopeptide monomers comprising at least 6 consecutive amino acids from theamino acid sequence as set forth in SEQ ID NO: 1, wherein the at leasttwo peptide monomers are each no longer than 30 amino acids, wherein themultimeric peptide is capable of reducing binding of PLIF to humanleukocytes under conditions which allow for the generation of activatedT cells that are cytotoxic to the pathogenic cells; and

(b) expanding the activated T cells, thereby generating the cytotoxic Tcell line.

According to an aspect of some embodiments of the present inventionthere is provided an isolated cytotoxic T cell line which comprises amultimeric peptide attached to an outer surface of T cells of the T cellline, the multimeric peptide comprising at least two peptide monomerslinked to one another, each of the at least two peptide monomerscomprising at least 6 consecutive amino acids from the amino acidsequence as set forth in SEQ ID NO: 1, wherein the at least two peptidemonomers are each no longer than 30 amino acids, wherein the multimericpeptide is capable of reducing binding of PLIF to human leukocytes.

According to an aspect of some embodiments of the present inventionthere is provided an isolated cytotoxic T cell line generated accordingto the methods described herein.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the cytotoxicT cell lines described herein.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a disease which is amenable totreatment by adoptive immunotherapy in a subject in need thereof, themethod comprising administering to the subject a therapeuticallyeffective amount of the cytotoxic T cell line described herein, therebytreating the disease.

According to an aspect of some embodiments of the present inventionthere is provided a cytotoxic T cell line generated according to themethod described herein for treating a disease caused by a pathogeniccell population amenable to cytotoxic T cell therapy.

According to an aspect of some embodiments of the present inventionthere is provided a bank comprising a plurality of the cytotoxic T celllines.

According to some embodiments of the invention, the method furthercomprises expanding the activated T cells.

According to some embodiments of the invention, the expanding iseffected using interleukin 2 (IL-2).

According to some embodiments of the invention, the pathogenic cellscomprise cancer cells.

According to some embodiments of the invention, the cancer cellscomprise breast cancer cells.

According to some embodiments of the invention, the breast cancer cellscomprise cells of the T47D or MCF-7 cell lines.

According to some embodiments of the invention, the T cells arecomprised in peripheral mononuclear blood cells (PBMCs).

According to some embodiments of the invention, the peptide is capableof increasing INF-γ secretion from activated leukocytes.

According to some embodiments of the invention, the peptide is a dimer.

According to some embodiments of the invention, each of the at least twopeptide monomers comprise no more than 15 consecutive amino acids fromthe amino acid sequence as set forth in SEQ ID NO: 1.

According to some embodiments of the invention, the at least twopeptides comprise an identical amino acid sequence.

According to some embodiments of the invention, each of the at least twopeptide monomers is attached to a Cysteine (Cys) residue.

According to some embodiments of the invention, the caboxy end of the atleast two peptide monomers is attached to the Cys residue.

According to some embodiments of the invention, each of the two peptidemonomers are attached via a non-peptide linker.

According to some embodiments of the invention, the at least two peptidemonomers are linked to one another by a disulfide bond.

According to some embodiments of the invention, the disulfide bond is anintermolecular disulfide bond formed between the Cys residues.

According to some embodiments of the invention, the multimeric peptidefurther comprises a Gly residue connecting the Cys residue to thecarboxy end of the at least two peptide monomers.

According to some embodiments of the invention, each of the two at leasttwo peptide monomers comprises the sequence selected from the groupconsisting of SEQ ID NOs: 2-7.

According to some embodiments of the invention, each of the at least twopeptide monomers consists of the sequence selected from the groupconsisting of SEQ ID NOs: 8-13.

According to some embodiments of the invention, the multimeric peptidecomprises at least one synthetic amino acid.

According to some embodiments of the invention, the at least two peptidemonomers comprises least three peptide monomers.

According to some embodiments of the invention, the at least two peptidemonomers are covalently linked to one another.

According to some embodiments of the invention, the disease is cancer.

According to some embodiments of the invention, the cancer of thesubject expresses at least one HLA class I allele which is identical toa HLA class I allele expressed on cancer cells used to generate thecytotoxic T cells.

According to some embodiments of the invention, when the cancer cellsare MCF-7, the cancer of the subject is selected from the groupconsisting of breast cancer, colon cancer, lung cancer and renal cancer.

According to some embodiments of the invention, the breast cancercomprises triple negative breast cancer.

According to some embodiments of the invention, the cytotoxic T cellline is generated using PBMCs.

According to some embodiments of the invention, the PBMCs are autologousto the subject.

According to some embodiments of the invention, the cancer of thesubject is selected from the group consisting of breast cancer, coloncancer, lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acutelymphatic leukemia (ALL) and renal cancer.

According to some embodiments of the invention, the breast cancercomprises triple negative breast cancer.

According to some embodiments of the invention, the cytotoxic T cellline is generated by incubating T cells with pathogenic cells in thepresence of a multimeric peptide comprising at least two peptidemonomers linked to one another, each of the at least two peptidemonomers comprising at least 6 consecutive amino acids from the aminoacid sequence as set forth in SEQ ID NO: 1, wherein the at least twopeptide monomers are each no longer than 30 amino acids, wherein themultimeric peptide is capable of reducing binding of PLIF to humanleukocytes under conditions which allow expansion of the T cells.

According to some embodiments of the invention, the pathogenic cells arederived from the subject.

According to some embodiments of the invention, the pathogenic cells arenot derived from the subject.

According to some embodiments of the invention, the cytotoxic T cellline is for treating a disease caused by a pathogenic cell populationamenable to cytotoxic T cell therapy.

According to some embodiments of the invention, the disease is cancer.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying images. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a flow chart illustrating the generation of specificanti-breast cancer cytotoxic T cell lines.

FIGS. 2A-2C are photomicrographs illustrating the effect of 5 dayincubation of C24D peptide on T47D tumor cytolysis in the presence ofperipheral blood mononuclear cells (PBMC).

FIGS. 3A-3C are photomicrographs illustrating the effect of 7 dayincubation of C24D peptide on T47D tumor cytolysis in the presence ofperipheral blood mononuclear cells (PBMC).

FIGS. 4A-4C are photomicrographs illustrating the effect of 5 dayincubation of C24D peptide on MCF7 tumor cytolysis in the presence ofperipheral blood mononuclear cells (PBMC).

FIGS. 5A-5C are photomicrographs illustrating the effect of 7 dayincubation of C24D peptide on MCF7 tumor cytolysis in the presence ofperipheral blood mononuclear cells (PBMC).

FIG. 6 is a bar graph illustrating the effect of C24D treatment on T47Dand MCF7 tumor cell cytolysis by PBMC in culture (6 days).

FIGS. 7A-7D are photomicrographs illustrating the effect of 1.5 hours ofculture of the anti-T47D cell line on T47D cells (FIG. 7B) and MCF7cells (FIG. 7D).

FIG. 8 is a graph illustrating the effect of 1.5 hours of culture of theanti-T47D cell line on T47D cells (blue) and MCF7 cells (pink).

FIGS. 9A-9B are graphs illustrating the results of FACS analysisanalyzing apoptotosis of T47D Tumor Cells by the Specific CTL. TumorCells were cultured with anti-T47D CTL, for 0 h and 2 h, at E:T ratio1:3. Cells were labeled with FITC Annexin V and PI.

FIG. 10 is a graph illustrating that anti-T47D CTL induced earlyapoptosis as estimated by measurement of annexin V.

FIGS. 11A-11D are photomicrographs of MDA-MB231 tumor cell cytolysis byanti-MCF7 CTL. A, C. Control MDA-MB231 target cells incubated withnon-CTL PBMC. B. Cytolysis following incubation with HLA-A*0201+identical anti-MCF7 CTL. D. No cytolysis with anti-HLA-A2-T47D CTL.

FIG. 12 is a graph illustrating the cross cytotoxicity of MDA-MB231 bydifferent anti-tumor CTL as measured by MTT viability assay. Note thecytotoxicity of MDA-MB231 target treated with anti-MCF7 CTL compared toanti-T47D CTL.

Each value represents the mean from triplicate wells of the 3experiments performed.Effector target ratio 1:1; 5 hours incubation at 37° C.

FIG. 13 is a graph illustrating the level of interferon-γ secretion byCTL following re-stimulation with MCF7, MDA-MB231 and T47D, indicatingantigenic cross reactivity. Effector target ratio 1:1; 5 hoursincubation at 37° C.

FIG. 14 is a graph illustrating interferon-γ secretion by anti-MCF7 CTLre-stimulated with MDA-MB231 and MCF-7. Effector target ratio 1:1 and1:3; 5 hours incubation at 37° C.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates togeneration of cytotoxic tumor specific cell lines using PlacentalImmunoregulatory Ferritin (PLIF) peptides.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Tumor-specific cytotoxic lymphocytes have been proposed as a therapeuticfor the treatment of cancer. They are usually expanded from peripheralblood mononuclear cells (PBMC) taken from tumor-bearing patients usingantigen-presenting cells pulsed with irradiated tumor cells, tumorpeptides, tumor lysates or fused tumor cells. This results in theexpansion of MHC class I-restricted cytotoxic T cell lines over severalweeks of culture. Tumor-specific cytotoxic T cell lines can also bederived as a subpopulation of tumor-infiltrating lymphocytes bymodifying the methodologies, including a purification step based on theselection of CD8 T cells.

In human clinical trials, infusion of tumor-specific T cells derivedfrom tumor-infiltrating lymphocytes or draining lymph nodes has shownlimited but encouraging clinical responses in specific settings.Unfortunately, the ability to expand tumor antigen-specific T cells exvivo from cancer patients is technically difficult due to numerousobstacles, including initiating cultures with low numbers oftumor-specific T cells and the physical inability to obtaintumor-infiltrating lymphocytes from patients with the most commonmalignancies.

Since Placenta Immunomodulatory Factor (PLIF) is a protein which isknown to induce immune tolerance to certain malignant cells, the presentinventors propose that an agent which blocks the activity of PLIF mayallow for the activation of tumor specific T cells.

Whilst reducing the present invention to practice the present inventorsshowed that it was possible to generate cytotoxic T cells by incubatingT cells with cancer cells in the presence of the PLIF antagonist C24D(FIGS. 2A-2C-FIGS. 5A-5C). Generation of T cell lines from theseactivated cells was effected using the expansion agent, interleukin 2.These cell lines were shown to be both cytotoxic and tumor specific(FIGS. 7A-7D).

The present inventors further showed that T cells activated using onetype of cancer cell were cytotoxic against another cancer cell providedthat they shared HLA class I alleles. More specifically, the presentinventors showed that T cells activated using the MCF7 breast cancercell line were cytotoxic against MDA-MB231 cells (a breast cancer triplenegative cell line (FIGS. 11A-11D and FIG. 12). Both of these breastcancer cell lines express HLA* 0201 peptide on their surface.

Accordingly, the present inventors propose the use of allogeneicHLA-A2-matched tumor cells as stimulator cells together with a PLIFantagonist for the generation of cytotoxic T cells, thereby expandingtheir potential for treating alloreactive tumors.

Thus, according to one aspect of the present invention there is providedan in-vitro method of activating T cells, the method comprisingincubating T cells with pathogenic cells in the presence of a multimericpeptide comprising at least two peptide monomers linked to one another,each of the at least two peptide monomers comprising at least 6consecutive amino acids from the amino acid sequence as set forth in SEQID NO: 1, wherein the at least two peptide monomers are each no longerthan 30 amino acids, wherein the multimeric peptide is capable ofreducing binding of PLIF to human leukocytes under conditions whichallow expansion of the T cells.

As used herein, the phrase “An in-vitro method” refers to a method thatdoes not occur within an animal. It will be appreciated that the term“in-vitro” may include the term “ex vivo”, wherein at least one of thecomponents of the incubation culture are derived from an animal.

The various components of the incubation reaction are discussed hereinbelow:

T Cells

The term “T cells” refers to cytotoxic T cells. Cytotoxic T cellstypically express a T cell receptor that binds to a specific antigen onthe target cell.

The T cells may be derived from any mammalian species, such as human andmay be obtained from white blood cell preparations or peripheral bloodmononuclear cells (PBMCs) derived from a subject.

Alternatively, the T cells may have migrated into the tumor (i.e. may becomprised in tumor-infiltrating lymphocytes). Tumor infiltratinglymphocytes (TILs) can be isolated from an individual (e.g. during atumor biopsy) and cultured in vitro (Kawakami, Y. et al. (1989) J.Immunol. 142: 2453-3461). An exemplary method for obtaining TILsincludes plating viable cells (e.g. 1×10⁶) of a single-cell suspensionof enzymatically digested explant of metastatic tumor. It will beappreciated that the TILs may be isolated from fresh tumors or fromfrozen tissue (at the cost of lower yield).

The T cells are typically comprised in a heterogeneous population ofwhite blood cells which includes antigen presenting cells andmacrophages.

As mentioned, the T cells may be derived from PBMCs. PBMCs may beprepared as follows: Buffy coats from blood bank donors are layered ontoLymphoprep solution (Nycomed, Oslo, Norway) and spun at 2000 rpm forabout 20 minutes. The interface layer is collected, washed, counted, andresuspended in PBS; pH 7.4 to the desired cell concentration.

Pathogenic Cells:

Contemplated pathogenic cells include any cells that comprise antigenicdeterminants on their surface. The pathogenic cells may be bacterial,fungal or viral. The pathogenic cells may be cells damaged byenvironmental factors such as ultraviolet light and other radiations.Alternatively, the pathogenic cells may be diseased cells such as cancercells.

According to one embodiment the pathogenic cells are associated with anupregulated amount of Placenta Immunomodulatory Factor (PLIF). Suchcells include lymphocytes from Hodgkin's and non-Hodgkin's lymphoma,acute lymphatic leukemia (ALL), cells from human breast cancer tissuesand cells from breast cancer cell lines (e.g. T47D and MCF-7).

As used herein, the term “upregulated” refers to an increased amount ofPLIF as compared to healthy cells of the same kind as the pathogeniccells. Thus, for example, if the pathogenic cells comprise lymphocytes,the amount of PLIF is upregulated as compared to the amount of PLIF inhealthy lymphoctyes. As another example, if the pathogenic cellscomprise breast cancer cells, the amount of PLIF is upregulated ascompared to the amount of PLIF in healthy breast tissue.

The pathogenic cells may be obtained from a patient (e.g. during abiopsy) or may be available as a cell line.

Multimeric Peptide

The phrase “multimeric peptide” as used herein, describes a peptideformed from two or more peptide monomers (i.e. two or more peptidechains) that are associated covalently or non-covalently, with orwithout linkers. It will be appreciated that the peptide monomers arenot linked together so as to form an amide bond through the amine groupof one monomer and the carboxylic acid group of the other monomer so asto form a single extended chain.

According to a particular embodiment, the multimeric peptide is a dimer(i.e. comprises two peptide monomers that are associated covalently ornon-covalently, with or without linkers). According to a particularembodiment, the two peptide monomers are not linked via a peptide bond.

The multimeric peptides disclosed herein are capable of blocking bindingof PLIF to its receptor on white blood cells, thereby acting as anantagonist to the endogenous activity of Placenta ImmunomodulatoryFactor (PLIF).

PLIF is a protein composed of 165 amino acids. Of these, 117 match theferritin heavy chain sequence, whereas the C-terminal 48 amino acids(C48) has a sequence which is not related to ferritin. It has been shownthat the subcloned recombinant C48 peptide exhibits the bioactivity andtherapeutic properties of PLIF [Moroz et al, J. Biol. Chem. 2002, 277,12901-12905].

Methods of ascertaining whether the peptides are capable of antagonizingPLIF are known in the art and include for example analyzing the amountof each peptide that is capable of binding to white blood cells(leukocytes) both separately and/or in the same culture.

Binding affinity can be measured by any assay known or available tothose skilled in the art, including but not limited to BIAcoremeasurements, ELISA assays, competition assays, etc. Bioactivity can bemeasured in vivo or in vitro by any assay known or available to thoseskilled in the art.

According to one embodiment, binding is measured using an antibody whichis capable of specifically recognizing the peptides disclosed herein(i.e. binds with a higher affinity to the multimeric peptides disclosedherein than for C48 (SEQ ID NO: 100) or PLIF under identicalconditions). Such antibodies are further described herein below.

The multimeric peptides of this aspect of the present inventiontypically comprise additional functions such as being capable ofincreasing interferon gamma (INF-γ) secretion and/or interleukin-10(IL-10) secretion from activated leukocytes. According to oneembodiment, secretion of INF-γ is increased by at least two fold, ormore preferably by at least five fold the amount of INF-γ that isbasally secreted from activated leukocytes (i.e. in the absence of thedisclosed peptides).

Methods of analyzing INF-γ secretion include but are not limited toELISA kits such as those available from DPC, and R&D Systems, USA.

In some embodiments, the multimeric peptide is such that the amino acidsequence of each of its monomers are the same, thus forming ahomomultimeric peptide. When the multimeric peptide is a dimer and thetwo monomers are identical, a homodimeric peptide is formed.

In some embodiments, the multimeric peptide is such that the amino acidsequence of at least two of its peptide monomers are different, thusforming a heteromultimeric peptide. When the multimeric peptide is adimer and the two monomers are different, a heterodimeric peptide isformed.

As mentioned, the monomers of the multimeric peptide of this aspect ofthe present invention are derived from the C terminal amino acids ofPlacenta Immunomodulatory Factor (PLIF) and include at least 6consecutive amino acids from the sequence as set forth in SEQ ID NO: 1(His-His-Leu-Leu-Arg-Pro-Arg-Arg-Lys-Arg-Pro-His-Ser-Ile-Pro-Thr-Pro-Ile-Leu-Ile-Phe-Arg-Ser-Pro).

According to some embodiments, each monomer of the multimeric peptidecomprises at least 7 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 8 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 9 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 10 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 11 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 12 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 13 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 14 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 15 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 16 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 17 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 18 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 19 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 20 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 21 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 22 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises at least 23 consecutive amino acids from the sequence as setforth in SEQ ID NO: 1.

According to some embodiments, each monomer of the multimeric peptidecomprises the full length sequence as set forth in SEQ ID NO: 1.

According to a particular embodiment the amino acid sequence derivedfrom SEQ ID NO: 1 is HSIPTPILIFRSP (SEQ ID NO: 2), HLLRPRRRKRPHSI (SEQID NO: 3), RPRRRKRPHSIP (SEQ ID NO: 4), SIPTPILIFRSP (SEQ ID NO: 5),PHSIPTPILIFRSP (SEQ ID NO: 6) or HHLLRPRRRKR (SEQ ID NO: 7).

Preferably, each monomer of the multimeric peptide comprises at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14 consecutive amino acids from the sequenceas set forth in SEQ ID NO: 14-RPHSIPTPILIFRSP.

Additional contemplated peptides include those set forth in Table 1,herein below.

TABLE 1 SEQ ID Sequence 15 His-His-Leu-Leu-Arg-Pro 16His-Leu-Leu-Arg-Pro-Arg 17 Leu-Leu-Arg-Pro-Arg-Arg 18Leu-Arg-Pro-Arg-Arg-Lys 19 Arg-Pro-Arg-Arg-Lys-Arg 20Pro-Arg-Arg-Lys-Arg-Pro 21 Arg-Arg-Lys-Arg-Pro-His 22Arg-Lys-Arg-Pro-His-Ser 23 Lys-Arg-Pro-His-Ser-Ile 24Arg-Pro-His-Ser-Ile-Pro 25 Pro-His-Ser-Ile-Pro-Thr 26His-Ser-Ile-Pro-Thr-Pro 27 Ser-Ile-Pro-Thr-Pro-Ile 28Ile-Pro-Thr-Pro-Ile-Leu 29 Pro-Thr-Pro-Ile-Leu-Ile 30Thr-Pro-Ile-Leu-Ile-Phe 31 Pro-Ile-Leu-Ile-Phe-Arg 32Ile-Leu-Ile-Phe-Arg-Ser 33 Leu-Ile-Phe-Arg-Ser-Pro 34His-His-Leu-Leu-Arg-Pro-Arg 35 His-Leu-Leu-Arg-Pro-Arg-Arg 36Leu-Leu-Arg-Pro-Arg-Arg-Lys 37 Leu-Arg-Pro-Arg-Arg-Lys-Arg 38Arg-Pro-Arg-Arg-Lys-Arg-Pro 39 Pro-Arg-Arg-Lys-Arg-Pro-His 40Arg-Arg-Lys-Arg-Pro-His-Ser 41 Arg-Lys-Arg-Pro-His-Ser-Ile 42Lys-Arg-Pro-His-Ser-Ile-Pro 43 Arg-Pro-His-Ser-Ile-Pro-Thr 44Pro-His-Ser-Ile-Pro-Thr-Pro 45 His-Ser-Ile-Pro-Thr-Pro-Ile 46Ser-Ile-Pro-Thr-Pro-Ile-Leu 47 Ile-Pro-Thr-Pro-Ile-Leu-Ile 48Pro-Thr-Pro-Ile-Leu-Ile-Phe 49 Thr-Pro-Ile-Leu-Ile-Phe-Arg 50Pro-Ile-Leu-Ile-Phe-Arg-Ser 51 Ile-Leu-Ile-Phe-Arg-Ser-Pro 52His-His-Leu-Leu-Arg-Pro-Arg-Arg 53 His-Leu-Leu-Arg-Pro-Arg-Arg-Lys 54Leu-Leu-Arg-Pro-Arg-Arg-Lys-Arg 55 Leu-Arg-Pro-Arg-Arg-Lys-Arg-Pro 56Arg-Pro-Arg-Arg-Lys-Arg-Pro-His 57 Pro-Arg-Arg-Lys-Arg-Pro-His-Ser 58Arg-Arg-Lys-Arg-Pro-His-Ser-Ile 59 Arg-Lys-Arg-Pro-His-Ser-Ile-Pro 60Lys-Arg-Pro-His-Ser-Ile-Pro-Thr 61 Arg-Pro-His-Ser-Ile-Pro-Thr-Pro 62Pro-His-Ser-Ile-Pro-Thr-Pro-Ile 63 His-Ser-Ile-Pro-Thr-Pro-Ile-Leu 64Ser-Ile-Pro-Thr-Pro-Ile-Leu-Ile 65 Ile-Pro-Thr-Pro-Ile-Leu-Ile-Phe 66Pro-Thr-Pro-Ile-Leu-Ile-Phe-Arg 67 Thr-Pro-Ile-Leu-Ile-Phe-Arg-Ser 68Pro-Ile-Leu-Ile-Phe-Arg-Ser-Pro 69 His-His-Leu-Leu-Arg-Pro-Arg-Arg-Lys70 His-Leu-Leu-Arg-Pro-Arg-Arg-Lys-Arg 71Leu-Leu-Arg-Pro-Arg-Arg-Lys-Arg-Pro 72Leu-Arg-Pro-Arg-Arg-Lys-Arg-Pro-His 73Arg-Pro-Arg-Arg-Lys-Arg-Pro-His-Ser 74Pro-Arg-Arg-Lys-Arg-Pro-His-Ser-Ile 75Arg-Arg-Lys-Arg-Pro-His-Ser-Ile-Pro 76Arg-Lys-Arg-Pro-His-Ser-Ile-Pro-Thr 77Lys-Arg-Pro-His-Ser-Ile-Pro-Thr-Pro 78Arg-Pro-His-Ser-Ile-Pro-Thr-Pro-Ile 79Pro-His-Ser-Ile-Pro-Thr-Pro-Ile-Leu 80His-Ser-Ile-Pro-Thr-Pro-Ile-Leu-Ile 81Ser-Ile-Pro-Thr-Pro-Ile-Leu-Ile-Phe 82Ile-Pro-Thr-Pro-Ile-Leu-Ile-Phe-Arg 83Pro-Thr-Pro-Ile-Leu-Ile-Phe-Arg-Ser 84Thr-Pro-Ile-Leu-Ile-Phe-Arg-Ser-Pro 85His-His-Leu-Leu-Arg-Pro-Arg-Arg-Lys-Arg 86His-Leu-Leu-Arg-Pro-Arg-Arg-Lys-Arg-Pro 87Leu-Leu-Arg-Pro-Arg-Arg-Lys-Arg-Pro-His 88Leu-Arg-Pro-Arg-Arg-Lys-Arg-Pro-His-Ser 89Arg-Pro-Arg-Arg-Lys-Arg-Pro-His-Ser-Ile 90Pro-Arg-Arg-Lys-Arg-Pro-His-Ser-Ile-Pro 91Arg-Arg-Lys-Arg-Pro-His-Ser-Ile-Pro-Thr 92Arg-Lys-Arg-Pro-His-Ser-Ile-Pro-Thr-Pro 93Lys-Arg-Pro-His-Ser-Ile-Pro-Thr-Pro-Ile 94Arg-Pro-His-Ser-Ile-Pro-Thr-Pro-Ile-Leu 95Pro-His-Ser-Ile-Pro-Thr-Pro-Ile-Leu-Ile 96His-Ser-Ile-Pro-Thr-Pro-Ile-Leu-Ile-Phe 97Ser-Ile-Pro-Thr-Pro-Ile-Leu-Ile-Phe-Arg 98Ile-Pro-Thr-Pro-Ile-Leu-Ile-Phe-Arg-Ser 99Pro-Thr-Pro-Ile-Leu-Ile-Phe-Arg-Ser-Pro

The term “peptide” as used herein refers to a polymer of natural orsynthetic amino acids, encompassing native peptides (either degradationproducts, synthetically synthesized peptides or recombinant peptides)and peptidomimetics (typically, synthetically synthesized peptides), aswell as peptoids and semipeptoids which are peptide analogs, which mayhave, for example, modifications rendering the peptides even more stablewhile in a body or more capable of penetrating into cells.

Such modifications include, but are not limited to N terminusmodification, C terminus modification, peptide bond modification,including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C—NH, CH2-O,CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications, and residuemodification. Methods for preparing peptidomimetic compounds are wellknown in the art and are specified, for example, in Quantitative DrugDesign, C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press(1992), which is incorporated by reference as if fully set forth herein.Further details in this respect are provided hereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH3)-CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(˜CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine(Nol), ring-methylated derivatives of Phe, halogenated derivatives ofPhe or o-methyl-Tyr.

In addition to the above, the peptides of the present invention may alsoinclude one or more modified amino acids or one or more non-amino acidmonomers (e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification and in the claims section below theterm “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids (stereoisomers).

Tables 2 and 3 below list naturally occurring amino acids (Table 2) andnon-conventional or modified amino acids (Table 3) which can be usedwith the present invention.

TABLE 2 One-letter Three-Letter Symbol Abbreviation Amino Acid A Alaalanine R Arg Arginine N Asn Asparagine D Asp Aspartic acid C CysCysteine Q Gln Glutamine E Glu Glutamic Acid G Gly glycine H HisHistidine I Iie isoleucine L Leu leucine K Lys Lysine M Met Methionine FPhe phenylalanine P Pro Proline S Ser Serine T Thr Threonine W Trptryptophan Y Tyr tyrosine V Val Valine X Xaa Any amino acid as above

TABLE 3 Code Non-conventional amino acid Code Non-conventional aminoacid Nmala L-N-methylalanine Abu α-aminobutyric acid NmargL-N-methylarginine Mgabu α-amino-α-methylbutyrate NmasnL-N-methylasparagine Cpro aminocyclopropane- Nmasp L-N-methylasparticacid carboxylate Nmcys L-N-methylcysteine Aib aminoisobutyric acid NmginL-N-methylglutamine Norb aminonorbornyl- Nmglu L-N-methylglutamic acidcarboxylate Nmhis L-N-methylhistidine Chexa cyclohexylalanine NmileL-N-methylisolleucine Cpen cyclopentylalanine Nmleu L-N-methylleucineDal D-alanine Nmlys L-N-methyllysine Darg D-arginine NmmetL-N-methylmethionine Dasp D-aspartic acid Nmnle L-N-methylnorleucineDcys D-cysteine Nmnva L-N-methylnorvaline Dgln D-glutamine NmornL-N-methylornithine Dglu D-glutamic acid Nmphe L-N-methylphenylalanineDhis D-histidine Nmpro L-N-methylproline Dile D-isoleucine NmserL-N-methylserine Dleu D-leucine Nmthr L-N-methylthreonine Dlys D-lysineNmtrp L-N-methyltryptophan Dmet D-methionine Nmtyr L-N-methyltyrosineDorn D-ornithine Nmval L-N-methylvaline Dphe D-phenylalanine NmetgL-N-methylethylglycine Dpro D-proline Nmtbug L-N-methyl-t-butylglycineDser D-serine Nle L-norleucine Dthr D-threonine Nva L-norvaline DtrpD-tryptophan Maib α-methyl-aminoisobutyrate Dtyr D-tyrosine Mgabuα-methyl-γ-aminobutyrate Dval D-valine Mchexa α ethylcyclohexylalanineDmala D-α-methylalanine Mcpen α-methylcyclopentylalanine DmargD-α-methylarginine Manap α-methyl-α-napthylalanine DmasnD-α-methylasparagine Mpen α-methylpenicillamine DmaspD-α-methylaspartate Nglu N-(4-aminobutyl)glycine DmcysD-α-methylcysteine Naeg N-(2-aminoethyl)glycine DmglnD-α-methylglutamine Norn N-(3-aminopropyl)glycine DmhisD-α-methylhistidine Nmaabu N-amino-α-methylbutyrate DmileD-α-methylisoleucine Anap α-napthylalanine Dmleu D-α-methylleucine NpheN-benzylglycine Dmlys D-α-methyllysine Ngln N-(2-carbamylethyl)glycineDmmet D-α-methylmethionine Nasn N-(carbamylmethyl)glycine DmornD-α-methylornithine Nglu N-(2-carboxyethyl)glycine DmpheD-α-methylphenylalanine Nasp N-(carboxymethyl)glycine DmproD-α-methylproline Ncbut N-cyclobutylglycine Dmser D-α-methylserine NchepN-cycloheptylglycine Dmthr D-α-methylthreonine Nchex N-cyclohexylglycineDmtrp D-α-methyltryptophan Ncdec N-cyclodecylglycine DmtyD-α-methyltyrosine Ncdod N-cyclododeclglycine Dmval D-α-methylvalineNcoct N-cyclooctylglycine Dnmala D-α-methylalnine NcproN-cyclopropylglycine Dnmarg D-α-methylarginine NcundN-cycloundecylglycine Dnmasn D-α-methylasparagine NbhmN-(2,2-diphenylethyl)glycine Dnmasp D-α-methylasparatate Nbhe N-(3,3-Dnmcys D-α-methylcysteine diphenylpropyl)glycine NhtrpN-(3-indolylyethyl)glycine Dnmleu D-N-methylleucine NmgabuN-methyl-γ-aminobutyrate Dnmlys D-N-methyllysine DnmmetD-N-methylmethionine Nmchexa N-methylcyclohexylalanine NmcpenN-methylcyclopentylalanine Dnmorn D-N-methylornithine DnmpheD-N-methylphenylalanine Nala N-methylglycine Dnmpro D-N-methylprolineNmaib N-methylaminoisobutyrate Dnmser D-N-methylserine NileN-(1-methylpropyl)glycine Dnmser D-N-methylserine NileN-(2-methylpropyl)glycine Dnmthr D-N-methylthreonine NleuN-(2-methylpropyl)glycine Nva N-(1-methylethyl)glycine DnmtrpD-N-methyltryptophan Nmanap N-methyla-napthylalanine DnmtyrD-N-methyltyrosine Nmpen N-methylpenicillamine Dnmval D-N-methylvalineNhtyr N-(p-hydroxyphenyl)glycine Gabu γ-aminobutyric acid NcysN-(thiomethyl)glycine Tbug L-t-butylglycine Pen penicillamine EtgL-ethylglycine Mala L-α-methylalanine Hphe L-homophenylalanine MasnL-α-methylasparagine Marg L-α-methylarginine MtbugL-α-methyl-t-butylglycine Masp L-α-methylaspartate MetgL-methylethylglycine Mcys L-α-methylcysteine Mglu L-α-methylglutamateMgln L-α thylglutamine Mhphe L-α- Mhis L-α-methylhistidinemethylhomophenylalanine Nmet N-(2-methylthioethyl)glycine MileL-α-methylisoleucine Narg N-(3-guanidinopropyl)glycine DnmglnD-N-methylglutamine Nthr N-(1-hydroxyethyl)glycine DnmgluD-N-methylglutamate Nser N-(hydroxyethyl)glycine DnmhisD-N-methylhistidine Nhis N-(imidazolylethyl)glycine DnmileD-N-methylisoleucine Nhtrp N-(3-indolylyethyl)glycine DnmleuD-N-methylleucine Nmgabu N-methyl-γ-aminobutyrate DnmlysD-N-methyllysine Dnmmet D-N-methylmethionine NmchexaN-methylcyclohexylalanine Nmcpen N-methylcyclopentylalanine DnmornD-N-methylornithine Dnmphe D-N-methylphenylalanine Nala N-methylglycineDnmpro D-N-methylproline Nmaib N-methylaminoisobutyrate DnmserD-N-methylserine Nile N-(1-methylpropyl)glycine DnmthrD-N-methylthreonine Nleu N-(2-methylpropyl)glycine NvalN-(1-methylethyl)glycine Dnmtrp D-N-methyltryptophan NmanapN-methyla-napthylalanine Dnmtyr D-N-methyltyrosine NmpenN-methylpenicillamine Dnmval D-N-methylvaline NhtyrN-(p-hydroxyphenyl)glycine Gabu γ-aminobutyric acid NcysN-(thiomethyl)glycine Tbug L-t-butylglycine Pen penicillamine EtgL-ethylglycine Mala L-α-methylalanine Hphe L-homophenylalanine MasnL-α-methylasparagine Marg L-α-methylarginine MtbugL-α-methyl-t-butylglycine Masp L-α-methylaspartate MetgL-methylethylglycine Mcys L-α-methylcysteine Mglu L-α-methylglutamateMgln L-α-methylglutamine Mhphe L-α- Mhis L-α-methylhistidinemethylhomophenylalanine Nmet N-(2-methylthioethyl)glycine MileL-α-methylisoleucine Mlys L-α-methyllysine Mleu L-α-methylleucine MnleL-α-methylnorleucine Mmet L-α-methylmethionine Morn L-α-methylornithineMnva L-α-methylnorvaline Mpro L-α-methylproline MpheL-α-methylphenylalanine Mthr L-α-methylthreonine mser L-α-methylserineMtyr L-α-methyltyrosine Mtrp L-α ethylvaline Nmhphe L-N- MvalL-α-methylleucine methylhomophenylalanine NnbhmN-(N-(3,3-diphenylpropyl) N-(N-(2,2-diphenylethyl) Nnbhecarbamylmethyl(1)glycine Nnbhm carbamylmethyl-glycine Nmbc1-carboxy-1-(2,2-diphenylethyl- amino)cyclopropane

It will be appreciated that additional peptides are contemplated by thepresent invention as well as those disclosed herein, which may besynthesized (comprising conservative or non-conservative substitutions)in order to “tweak” the peptides and generate PLIF-derived peptides withimproved characteristics i.e. comprising an enhanced ability to blockPLIF binding and/or to stimulate the secretion of IFN from Tlymphocytes.

Thus, in other embodiments, the peptide monomers comprise a homolog, avariant, or a functional fragment of the sequences described hereinabove. In another embodiment, the peptide monomers comprise an aminoacid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%identical to the sequences described herein above.

The term “conservative substitution” as used herein, refers to thereplacement of an amino acid present in the native sequence in thepeptide with a naturally or non-naturally occurring amino or apeptidomimetics having similar steric properties. Where the side-chainof the native amino acid to be replaced is either polar or hydrophobic,the conservative substitution should be with a naturally occurring aminoacid, a non-naturally occurring amino acid or with a peptidomimeticmoiety which is also polar or hydrophobic (in addition to having thesame steric properties as the side-chain of the replaced amino acid).

As naturally occurring amino acids are typically grouped according totheir properties, conservative substitutions by naturally occurringamino acids can be easily determined bearing in mind the fact that inaccordance with the invention replacement of charged amino acids bysterically similar non-charged amino acids are considered asconservative substitutions.

For producing conservative substitutions by non-naturally occurringamino acids it is also possible to use amino acid analogs (syntheticamino acids) well known in the art. A peptidomimetic of the naturallyoccurring amino acid is well documented in the literature known to theskilled practitioner.

When affecting conservative substitutions the substituting amino acidshould have the same or a similar functional group in the side chain asthe original amino acid.

The phrase “non-conservative substitutions” as used herein refers toreplacement of the amino acid as present in the parent sequence byanother naturally or non-naturally occurring amino acid, havingdifferent electrochemical and/or steric properties. Thus, the side chainof the substituting amino acid can be significantly larger (or smaller)than the side chain of the native amino acid being substituted and/orcan have functional groups with significantly different electronicproperties than the amino acid being substituted. Examples ofnon-conservative substitutions of this type include the substitution ofphenylalanine or cycohexylmethyl glycine for alanine, isoleucine forglycine, or —NH—CH[(—CH₂)₅—COOH]—CO— for aspartic acid. Thosenon-conservative substitutions which fall under the scope of the presentinvention are those which still constitute a peptide havinganti-bacterial properties.

The N and C termini of the peptides of the present invention may beprotected by function groups. Suitable functional groups are describedin Green and Wuts, “Protecting Groups in Organic Synthesis”, John Wileyand Sons, Chapters 5 and 7, 1991, the teachings of which areincorporated herein by reference.

Hydroxyl protecting groups include esters, carbonates and carbamateprotecting groups. Amine protecting groups include alkoxy and aryloxycarbonyl groups, as described above for N-terminal protecting groups.Carboxylic acid protecting groups include aliphatic, benzylic and arylesters, as described above for C-terminal protecting groups. In oneembodiment, the carboxylic acid group in the side chain of one or moreglutamic acid or aspartic acid residue in a peptide of the presentinvention is protected, preferably with a methyl, ethyl, benzyl orsubstituted benzyl ester.

Examples of N-terminal protecting groups include acyl groups (—CO—R1)and alkoxy carbonyl or aryloxy carbonyl groups (—CO—O—R1), wherein R1 isan aliphatic, substituted aliphatic, benzyl, substituted benzyl,aromatic or a substituted aromatic group. Specific examples of acylgroups include acetyl, (ethyl)-CO—, n-propyl-CO—, iso-propyl-CO—,n-butyl-CO—, sec-butyl-CO—, t-butyl-CO—, hexyl, lauroyl, palmitoyl,myristoyl, stearyl, oleoyl phenyl-CO—, substituted phenyl-CO—,benzyl-CO— and (substituted benzyl)-CO—. Examples of alkoxy carbonyl andaryloxy carbonyl groups include CH3-O—CO—, (ethyl)-O—CO—,n-propyl-O—CO—, iso-propyl-O—CO—, n-butyl-O—CO—, sec-butyl-O—CO—,t-butyl-O—CO—, phenyl-O— CO—, substituted phenyl-O—CO— and benzyl-O—CO—,(substituted benzyl)-O—CO—. Adamantan, naphtalen, myristoleyl, tuluen,biphenyl, cinnamoyl, nitrobenzoy, toluoyl, furoyl, benzoyl, cyclohexane,norbornane, Z-caproic. In order to facilitate the N-acylation, one tofour glycine residues can be present in the N-terminus of the molecule.

The carboxyl group at the C-terminus of the compound can be protected,for example, by an amide (i.e., the hydroxyl group at the C-terminus isreplaced with —NH₂, —NHR₂ and —NR₂R₃) or ester (i.e. the hydroxyl groupat the C-terminus is replaced with —OR₂). R₂ and R₃ are independently analiphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or asubstituted aryl group. In addition, taken together with the nitrogenatom, R₂ and R₃ can form a C4 to C8 heterocyclic ring with from about0-2 additional heteroatoms such as nitrogen, oxygen or sulfur. Examplesof suitable heterocyclic rings include piperidinyl, pyrrolidinyl,morpholino, thiomorpholino or piperazinyl. Examples of C-terminalprotecting groups include —NH₂, —NHCH₃, —N(CH₃)₂, —NH(ethyl),—N(ethyl)₂, —N(methyl) (ethyl), —NH(benzyl), —N(C1-C4 alkyl)(benzyl),—NH(phenyl), —N(C1-C4 alkyl) (phenyl), —OCH₃, —O-(ethyl), —O-(n-propyl),—O-(n-butyl), —O-(iso-propyl), —O-(sec-butyl), —O-(t-butyl), —O-benzyland —O-phenyl.

The peptides of the present invention may also comprise non-amino acidmoieties, such as for example, hydrophobic moieties (various linear,branched, cyclic, polycyclic or hetrocyclic hydrocarbons and hydrocarbonderivatives) attached to the peptides; various protecting groups,especially where the compound is linear, which are attached to thecompound's terminals to decrease degradation. Chemical (non-amino acid)groups present in the compound may be included in order to improvevarious physiological properties such; decreased degradation orclearance; decreased repulsion by various cellular pumps, improveimmunogenic activities, improve various modes of administration (such asattachment of various sequences which allow penetration through variousbathers, through the gut, etc.); increased specificity, increasedaffinity, decreased toxicity and the like.

Exemplary side chain protecting groups and their positioning aredescribed in the Examples section herein below.

Linking of the monomers of the PLIF derived monomers may be effectedusing any method known in the art provided that the linking does notsubstantially interfere with the bioactivity of the multimericpeptide—e.g. to interfere with the ability of the multimeric peptide toblock the binding of PLIF to receptors on leukocytes (e.g. T cells).

The monomers of this aspect of the present invention may be linkedthrough a linking moiety.

Examples of linking moieties include but are not limited to a simplecovalent bond, a flexible peptide linker, a disulfide bridge or apolymer such as polyethylene glycol (PEG). Peptide linkers may beentirely artificial (e.g., comprising 2 to 20 amino acid residuesindependently selected from the group consisting of glycine, serine,asparagine, threonine and alanine) or adopted from naturally occurringproteins. Disulfide bridge formation can be achieved, e.g., by additionof cysteine residues, as further described herein below. Linking throughpolyethylene glycols (PEG) can be achieved by reaction of monomershaving free cysteines with multifunctional PEGs, such as linearbis-maleimide PEGs. Alternatively, linking can be performed though theglycans on the monomer after their oxidation to aldehyde form and usingmultifunctional PEGs containing aldehyde-reactive groups.

Selection of the position of the link between the two monomers shouldtake into account that the link should not substantially interfere withthe ability of the multimer to block the binding of PLIF to receptors onT cells.

Thus, for example, the linking moiety is optionally a moiety which iscovalently attached to a side chain, an N-terminus or a C-terminus ofthe first peptide monomer, as well as to a side chain, an N-terminus ora C-terminus of the second peptide monomer.

Preferably the linking moiety is attached to the C-terminus of the firstpeptide monomer, and to the C-terminus of the second peptide monomer.

As mentioned, the linking moiety used in this aspect of the presentinvention may be a cysteine residue.

Thus, in some embodiments of the invention, each of the peptide monomerscomprises an amino acid sequence as described herein above and furthercomprise at least one cysteine residue, such that the peptide monomersare covalently linked to one another via a disulfide bridge formedbetween a cysteine residue in one peptide monomer and a cysteine residuein another peptide monomer.

Typically, the cysteine is situated at the carboxy end of the peptidemonomers.

Hereinthroughout, the phrases “disulfide bridge” and “disulfide bond”are used interchangeably, and describe a —S—S— bond.

The linker may comprise additional amino acids linked together bypeptide bonds which serve as spacers such that the linker does notinterfere with the biological activity of the final compound. The linkeris preferably made up of amino acids linked together by peptide bonds.Thus, in preferred embodiments, the linker is made up of from 1 to 10amino acids linked by peptide bonds, wherein the amino acids areselected from the 20 naturally occurring amino acids. Some of theseamino acids may be glycosylated, as is well understood by those in theart. In a more preferred embodiment, besides cysteine the amino acids inthe linker are selected from glycine, alanine, proline, asparagine,glutamine, and lysine. Even more preferably, besides cysteine, thelinker is made up of a majority of amino acids that are stericallyunhindered, such as glycine and alanine.

Thus, according to one embodiment the linker comprises the sequencecysteine-glycine.

Exemplary monomer sequences are thus set forth by the followingsequences:

CGHSIPTPILIFRSP, (SEQ ID NO: 8) CGHLLRPRRRKRPHSI, (SEQ ID NO: 9)CGRPRRRKRPHSIP, (SEQ ID NO: 10) CGSIPTPILIFRSP, (SEQ ID NO: 11)CGPHSIPTPILIFRSP (SEQ ID NO: 12) or CGHHLLRPRRRKR. (SEQ ID NO: 13)

Non-peptide linkers are also possible. For example, alkyl linkers suchas —NH— (CH.sub.2).sub.s-C(O)—, wherein s=2-20 could be used. Thesealkyl linkers may further be substituted by any non-sterically hinderinggroup such as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl,Br), CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEGlinker.

Thus, in some embodiments, at least one of monomers is PEGylated orchemically modified to another form. PEGylation of the molecules can becarried out, e.g., according to the methods described in Youngster etal., Curr Pharm Des (2002), 8:2139; Grace et al., J Interferon CytokineRes (2001), 21: 1103; Pepinsky et al., J Pharmacol Exp Ther (2001),297:1059; Pettit et al., J Biol Chem (1997), 272:2312; Goodson et al.Biotechnology NY (1990), 8:343; Katre; J Immunol (1990), 144:209,Behrens et al US2006/0198819 A1, Klausen et al US2005/0113565 A1.

Any kind of polyethylene glycol is suitable for the present inventionprovided that the PEG-polypeptide-oligomer is still capable ofantagonizing or neutralizing the binding of PLIF with its receptor whichcan be assayed according to methods known in the art.

Preferably, the polyethylene glycol of the polypeptide-dimer of thepresent invention is PEG 1000, 2000, 3000, 5000, 10000, 15000, 20000 or40000 with PEG 20000 or 40000 being particularly preferred.

According to another embodiment the link is effected using a couplingagent.

The term “coupling agent”, as used herein, refers to a reagent that cancatalyze or form a bond between two or more functional groupsintra-molecularly, inter-molecularly or both. Coupling agents are widelyused to increase polymeric networks and promote crosslinking betweenpolymeric chains, hence, in the context of some embodiments of thepresent invention, the coupling agent is such that can promotecrosslinking between polymeric chains; or such that can promotecrosslinking between amino functional groups and carboxylic functionalgroups, or between other chemically compatible functional groups ofpolymeric chains. In some embodiments of the present invention the term“coupling agent” may be replaced with the term “crosslinking agent”. Insome embodiments, one of the polymers serves as the coupling agent andacts as a crosslinking polymer.

By “chemically compatible” it is meant that two or more types offunctional groups can react with one another so as to form a bond.

Exemplary functional groups which are typically present in gelatins andalginates include, but are not limited to, amines (mostly primary amines—NH₂), carboxyls (—CO₂H), sulfhydryls and hydroxyls (—SH and —OHrespectively), and carbonyls (—COH aldehydes and —CO— ketones).

Primary amines occur at the N-terminus of polypeptide chains (called thealpha-amine), at the side chain of lysine (Lys, K) residues (theepsilon-amine), as found in gelatin, as well as in various naturallyoccurring polysaccharides and aminoglycosides. Because of its positivecharge at physiologic conditions, primary amines are usuallyoutward-facing (i.e., found on the outer surface) of proteins and othermacromolecules; thus, they are usually accessible for conjugation.

Carboxyls occur at the C-terminus of polypeptide chain, at the sidechains of aspartic acid (Asp, D) and glutamic acid (Glu, E), as well asin naturally occurring aminoglycosides and polysaccharides such asalginate. Like primary amines, carboxyls are usually on the surface oflarge polymeric compounds such as proteins and polysaccharides.

Sulfhydryls and hydroxyls occur in the side chain of cysteine (Cys, C)and serine, (Ser, S) respectively. Hydroxyls are abundant inpolysaccharides and aminoglycosides.

Carbonyls as ketones or aldehydes can be form in glycoproteins,glycosides and polysaccharides by various oxidizing processes, syntheticand/or natural.

According to some embodiments of the present invention, the couplingagent can be selected according to the type of functional groups and thenature of the crosslinking bond that can be formed therebetween. Forexample, carboxyl coupling directly to an amine can be afforded using acarbodiimide type coupling agent, such as EDC; amines may be coupled tocarboxyls, carbonyls and other reactive functional groups byN-hydroxysuccinimide esters (NHS-esters), imidoester, PFP-ester orhydroxymethyl phosphine; sulfhydryls may be coupled to carboxyls,carbonyls, amines and other reactive functional groups by maleimide,haloacetyl (bromo- or iodo-), pyridyldisulfide and vinyl sulfone;aldehydes as in oxidized carbohydrates, may be coupled to other reactivefunctional groups with hydrazide; and hydroxyl may be coupled tocarboxyls, carbonyls, amines and other reactive functional groups withisocyanate.

Hence, suitable coupling agents that can be used in some embodiments ofthe present invention include, but are not limited to, carbodiimides,NHS-esters, imidoesters, PFP-esters or hydroxymethyl phosphines.

The peptides of the present invention can be biochemically synthesizedsuch as by using standard solid phase techniques. These methods includeexclusive solid phase synthesis, partial solid phase synthesis methods,fragment condensation, classical solution synthesis. Solid phasepolypeptide synthesis procedures are well known in the art and furtherdescribed by John Morrow Stewart and Janis Dillaha Young, Solid PhasePolypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Synthetic peptides can be purified by preparative high performanceliquid chromatography [Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.] and the composition ofwhich can be confirmed via amino acid sequencing.

Recombinant techniques may also be used to generate the monomers of thepresent invention. To produce a peptide of the present invention usingrecombinant technology, a polynucleotide encoding the monomer of thepresent invention is ligated into a nucleic acid expression vector,which comprises the polynucleotide sequence under the transcriptionalcontrol of a cis-regulatory sequence (e.g., promoter sequence) suitablefor directing constitutive, tissue specific or inducible transcriptionof the monomers of the present invention in the host cells.

In addition to being synthesizable in host cells, the monomers of thepresent invention can also be synthesized using in vitro expressionsystems. These methods are well known in the art and the components ofthe system are commercially available.

Typically, the monomers are synthesized as individual peptides,following which, depending on the linking moiety present in themonomers, linking is effected. For example, if the linking moiety is acysteine residue, thiol oxidation is performed.

Thus, according to another aspect of the present invention there isprovided a method of generating a dimeric peptide, the method comprisinglinking two isolated peptides, which each of the at least two isolatedpeptides comprise at least 6 consecutive amino acids and no more than 30amino acids from the amino acid sequence as set forth in SEQ ID NO: 1.

When Cys residue is used as a linking moiety, disulfide bonds may beformed by oxidation thereof. In one embodiment the control of cysteinebond formation is exercised by choosing an oxidizing agent of the typeand concentration effective to optimize formation of the multimer.Examples of oxidizing agent include iodine, dimethylsulfoxide (DMSO),potassium ferricyanide, and the like.

If the monomers comprise two or more cysteine residues, isomersresulting from disulfide bonds of different binding manner may beerroneously obtained. A peptide dimer wherein a disulfide bond is formedbetween intended cysteine residues can be prepared by selecting aparticular combination of protecting groups for cysteine side chains.Examples of the combination of protecting groups include MeBz1(methylbenzyl) and Acm (acetamidemethyl) groups, Trt (trityl) and Acmgroups, Npys (3-nitro-2-pyridylthio) and Acm groups, S-Bu-t(S-tert-butyl) and Acm groups, and the like. For example, in the case ofa combination of MeBz1 and Acm groups, the preparation can be carriedout by a method comprising removing protecting groups other than MeBz1group and a protecting group(s) on the cysteine side chain, andsubjecting the resulting monomer solution to air-oxidation to form adisulfide bond(s) between the deprotected cysteine residues, followed bydeprotection and oxidization with iodine to form a disulfide bond(s)between the cysteine residues previously protected by Acm.

In embodiments where a peptide dimer is dimerized via a linker moiety,the linker may be incorporated into the peptide during peptidesynthesis. For example, where a linker moiety contains two functionalgroups capable of serving as initiation sites for peptide synthesis anda third functional group (e.g., a carboxyl group or an amino group) thatenables binding to another molecular moiety, the linker may beconjugated to a solid support. Thereafter, two peptide monomers may besynthesized directly onto the two reactive nitrogen groups of the linkermoiety in a variation of the solid phase synthesis technique.

In alternate embodiments where a peptide dimer is dimerized by a linkermoiety, the linker may be conjugated to the two peptide monomers of apeptide dimer after peptide synthesis. Such conjugation may be achievedby methods well established in the art. In one embodiment, the linkercontains at least two functional groups suitable for attachment to thetarget functional groups of the synthesized peptide monomers. Forexample, a linker with two free amine groups may be reacted with theC-terminal carboxyl groups of each of two peptide monomers. In anotherexample, linkers containing two carboxyl groups, either preactivated orin the presence of a suitable coupling reagent, may be reacted with theN-terminal or side chain amine groups, or C-terminal lysine amides, ofeach of two peptide monomers.

Monomers of the invention can be attached to water-soluble polymers(e.g., PEG) using any of a variety of chemistries to link thewater-soluble polymer(s) to the receptor-binding portion of the molecule(e.g., peptide+spacer). A typical embodiment employs a single attachmentjunction for covalent attachment of the water soluble polymer(s) to thereceptor-binding portion, however in alternative embodiments multipleattachment junctions may be used, including further variations whereindifferent species of water-soluble polymer are attached to thereceptor-binding portion at distinct attachment junctions, which mayinclude covalent attachment junction(s) to the spacer and/or to one orboth peptide chains. In some embodiments, the dimer or higher ordermultimer will comprise distinct species of peptide chain (i.e., aheterodimer or other heteromultimer). By way of example and notlimitation, a dimer may comprise a first peptide chain having a PEGattachment junction and the second peptide chain may either lack a PEGattachment junction or utilize a different linkage chemistry than thefirst peptide chain and in some variations the spacer may contain orlack a PEG attachment junction and the spacer, if PEGylated, may utilizea linkage chemistry different than that of the first and/or secondpeptide chains. An alternative embodiment employs a PEG attached to thespacer portion of the receptor-binding portion and a differentwater-soluble polymer (e.g., a carbohydrate) conjugated to a side chainof one of the amino acids of the peptide portion of the molecule.

The peptides of the present invention may also comprise non-amino acidmoieties, such as for example, hydrophobic moieties (various linear,branched, cyclic, polycyclic or heterocyclic hydrocarbons andhydrocarbon derivatives) attached to the peptides; various protectinggroups, especially where the compound is linear, which are attached tothe compound's terminals to decrease degradation. Chemical (non-aminoacid) groups present in the compound may be included in order to improvevarious physiological properties such; decreased degradation orclearance; decreased repulsion by various cellular pumps, improveimmunogenic activities, improve various modes of administration (such asattachment of various sequences which allow penetration through variousbathers, through the gut, etc.); increased specificity, increasedaffinity, decreased toxicity and the like.

According to one embodiment, the peptides of the present invention areattached to a sustained-release enhancing agent. Exemplarysustained-release enhancing agents include, but are not limited tohyaluronic acid (HA), alginic acid (AA), polyhydroxyethyl methacrylate(Poly-HEMA), polyethylene glycol (PEG), glyme andpolyisopropylacrylamide.

Attaching the amino acid sequence component of the peptides of theinvention to other non-amino acid agents may be by covalent linking, bynon-covalent complexion, for example, by complexion to a hydrophobicpolymer, which can be degraded or cleaved producing a compound capableof sustained release; by entrapping the amino acid part of the peptidein liposomes or micelles to produce the final peptide of the invention.The association may be by the entrapment of the amino acid sequencewithin the other component (liposome, micelle) or the impregnation ofthe amino acid sequence within a polymer to produce the final peptide ofthe invention.

Incubation Reaction

As mentioned, the method of this aspect of the present inventioncomprises incubating the white blood cell population (which comprises Tcells and antigen presenting cells) with pathogenic cells in thepresence of the multimeric peptide described above under conditionswhich allow activation and expansion of the T cells.

The phrase “activation of the T cells” refers to the induction of acytotoxic activity in the T cells.

Preferably, the white blood cell population is incubated with thepathogenic cells and the multimeric peptide for at least one day, morepreferably at least two days, three days, four days, five days, sixdays, seven days or more so as to ensure activation.

Additional agents may be included in the incubation including forexample serum (e.g. fetal calf serum) or serum replacements. The peptidemay be added throughout the incubation period or at one or two dayintervals.

Preferably, the cells are cultured together with the peptide underconditions that ensure survival or propagation of the T cells. Suchconditions include incubating at appropriate temperatures and pressureand in a medium that ensures cell survival. Exemplary media include RPMIor RPMI 1640 or AIM V.

The present invention contemplates expanding the T cells concomitantlywith the activation and/or following the activation.

Expansion of T-cell cultures can be accomplished by any of a number ofmethods as are known in the arts. For example, T cells may be expandedutilizing non-specific T-cell receptor stimulation in the presence offeeder lymphocytes and either IL-2 or IL-15. The non-specific T-cellreceptor stimulus can consist of around 30 ng/ml of OKT3, a mousemonoclonal anti-CD3 antibody available from Ortho, Raritan, N.J.

The T-cells may be modified to express a T-cell growth factor thatpromotes the growth and activation thereof. Any suitable methods ofmodification may be used. See, e.g., Sambrook and Russell, MolecularCloning, 3^(rd) ed., SCHL Press (2001). Desirably, modified T-cellsexpress the T-cell growth factor at high levels. T-cell growth factorcoding sequences, such as that of IL-2, are readily available in theart, as are promoters, the operable linkage of which to a T-cell growthfactor coding sequence promote high-level expression.

Following generation of cytotoxic T cells, they may be isolated togenerate a homogeneous population of isolated cytotoxic T cells.

Methods of isolating cytotoxic T cells from a mixed population of cellsare known in the art and include for example isolating T cells based onthe expression of a cell surface antigens such as CD8. This may beperformed using flow cytometry. A multitude of flow cytometers arecommercially available including for e.g. Becton Dickinson FACScan andFACScaliber (BD Biosciences, Mountain View, Calif.). Antibodies that maybe used for FACS analysis are taught in Schlossman S, Boumell L, et al,[Leucocyte Typing V. New York: Oxford University Press; 1995] and arewidely commercially available.

Additionally, or alternatively, a substrate including an antibody or aligand capable of specifically binding cell surface markers present on“harmful” or non-relevant cells, can be used to effectively depletethese cells from the mixed population of cells.

The affinity substrate according to the present invention can be acolumn matrix such as, for example agarose, cellulose and the like, orbeads such as, for example, magnetic beads onto which the antibodiesdescribed above, are immobilized.

Using the methods described above cytotoxic T cells and T cell lines maybe obtained.

Thus, according to another aspect of the present invention there isprovided a cytotoxic T cell line which comprises a multimeric peptideattached to an outer surface of T cells of the T cell line, themultimeric peptide comprising at least two peptide monomers linked toone another, each of the at least two peptide monomers comprising atleast 6 consecutive amino acids from the amino acid sequence as setforth in SEQ ID NO: 1, wherein the at least two peptide monomers areeach no longer than 30 amino acids, wherein the multimeric peptide iscapable of reducing binding of PLIF to human leukocytes.

The present invention contemplates a T cell line wherein the multimericpeptide described herein above binds to at least 5% of the cells, atleast 10% of the cells, at least 15% of the cells, at least 20% of thecells, at least 25% of the cells, at least 30% of the cells, at least35% of the cells, at least 40% of the cells, at least 45% of the cells,at least 50% of the cells, at least 55% of the cells, at least 60% ofthe cells, at least 65% of the cells, at least 70% of the cells, atleast 75% of the cells, at least 80% of the cells, at least 85% of thecells, at least 90% of the cells, at least 95% of the cells, at least99% of the cells, or even to 100% of the cells.

Exemplary methods of assaying activities of T cell lines include ⁵¹CRrelease cytotoxicity assays (Cerundolo, V. et al. (1990) Nature345:449-452) or lymphokine assays such as IFN-γ or TNF secretion assays[Schwartzentruber, D. et al., (1991) J. of Immunology 146:3674-3681].

The T cell lines described herein may be used for treating subjectshaving diseases which are amenable to treatment by adoptiveimmunotherapy (e.g. cancer, autoimmune diseases, HIV, hepatitis, HHV6,chronic fatigue syndrome).

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

As used herein, the phrase “subject in need thereof” refers to a subjectwhich has the disease. The subject may be a mammal, e.g. a human. Forexample if the disease being treated is breast cancer, the subject istypically one being diagnosed with breast cancer, with or withoutmetastasis, at any stage of the disease (e.g. IA, IB, IIA, IIB, IIC,IIIA, IIIB, IIIC or IV).

The T cell lines may be used immediately following generation or may bestored (e.g. frozen) and used when needed.

Exemplary cancers which may be treated using the T cell lines describedherein include, but are not limited to, adrenocortical carcinoma,hereditary; bladder cancer; breast cancer; breast cancer, ductal; breastcancer, invasive intraductal; breast cancer, sporadic; breast cancer,susceptibility to; breast cancer, type 4; breast cancer, type 4; breastcancer-1; breast cancer-3; breast-ovarian cancer; Burkitt's lymphoma;cervical carcinoma; colorectal adenoma; colorectal cancer; colorectalcancer, hereditary nonpolyposis, type 1; colorectal cancer, hereditarynonpolyposis, type 2; colorectal cancer, hereditary nonpolyposis, type3; colorectal cancer, hereditary nonpolyposis, type 6; colorectalcancer, hereditary nonpolyposis, type 7; dermatofibrosarcomaprotuberans; endometrial carcinoma; esophageal cancer; gastric cancer,fibrosarcoma, glioblastoma multiforme; glomus tumors, multiple;hepatoblastoma; hepatocellular cancer; hepatocellular carcinoma;leukemia, acute lymphoblastic; leukemia, acute myeloid; leukemia, acutemyeloid, with eosinophilia; leukemia, acute nonlymphocytic; leukemia,chronic myeloid; Li-Fraumeni syndrome; liposarcoma, lung cancer; lungcancer, small cell; lymphoma, non-Hodgkin's; lynch cancer familysyndrome II; male germ cell tumor; mast cell leukemia; medullarythyroid; medulloblastoma; melanoma, meningioma; multiple endocrineneoplasia; myeloid malignancy, predisposition to; myxosarcoma,neuroblastoma; osteosarcoma; ovarian cancer; ovarian cancer, serous;ovarian carcinoma; ovarian sex cord tumors; pancreatic cancer;pancreatic endocrine tumors; paraganglioma, familial nonchromaffin;pilomatricoma; pituitary tumor, invasive; prostate adenocarcinoma;prostate cancer; renal cell carcinoma, papillary, familial and sporadic;retinoblastoma; rhabdoid predisposition syndrome, familial; rhabdoidtumors; rhabdomyosarcoma; small-cell cancer of lung; soft tissuesarcoma, squamous cell carcinoma, head and neck; T-cell acutelymphoblastic leukemia; Turcot syndrome with glioblastoma; tylosis withesophageal cancer; uterine cervix carcinoma, Wilms' tumor, type 2; andWilms' tumor, type 1, etc.

Preferably, the cancer is breast cancer, melanoma, lung carcinoma, coloncancer, prostate cancer, ovarian carcinoma, renal cell carcinoma,glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphaticleukemia (ALL) and the like. The cancer may be metastatic ornon-metastatic.

According to a particular embodiment, the cancer is breast cancer (e.g.triple negative breast cancer).

It will be appreciated that preparation of cytotoxic T cell lines fortreatment of a disease in a particular subject may be effected usingcomponents which are autologous to that subject. Thus, for example, thepresent invention contemplates using T cells retrieved from the patientfor generating the T cell line. Additionally and/or alternatively thepathogenic cells used to stimulate the T cells may be autologous to thesubject.

The present inventors have shown that as long as the pathogenic cellsused to activate the T cells share at least one HLA class I allele withthe pathogenic cells present in the subject the generated T cell lineswill be cytotoxic and effective at treating the disease in the subject.

Thus, the pathogenic cells used to stimulate the T cells are preferablyallogeneic with the pathogenic cells in the subject. Verdegaal et al.,Human Immunology 60, 1196-1206, 1999, the contents of which areincorporated by reference herein teaches various tumors which share HLAclass I alleles.

Thus, the present invention contemplates activating T cells with breastcancer cells and using the activated T cells for treating renal cellcarcinoma, colon cancer, renal cancer and/or melanoma.

In addition, the present invention contemplates activating T cells withone type of breast cancer cells and using the activated T cells fortreating another type of breast cancer (as long as the cancers share HLAclass I alleles). For example, the present inventors have shown thatactivating T cells with the MCF7 breast cancer cell line generates Tcells lines which are cytotoxic against MDA-MB231 cells (a breast cancertriple negative cell line.

The T cell lines may be provided per se or as part of a pharmaceuticalcomposition.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the T cells accountablefor the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not abrogate the biologicalactivity and properties of the administered compound. The carrier mayalso include biological or chemical substances that modulate the immuneresponse.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

The T-cells can be administered by any suitable route as known in theart. For example, the T-cells may be administered as an intra-arterialor intravenous infusion, which preferably lasts approximately 30-60minutes. Other examples of routes of administration includeintraperitoneal, intrathecal and intralymphatic.

A suitable dose of T-cells to be administered is from about 2.3×10¹⁰T-cells to about 13.7×10¹⁰ T-cells.

According to one embodiment, the T cells are administered to the subjecttogether with the C24D peptide.

Additionally, or alternatively, the T cells are administered to thesubject with a T-cell growth factor. The T-cell growth factor can be anysuitable growth factor that promotes the growth and activation of theT-cells administered. Examples of suitable T-cell growth factors includeIL-2, IL-7 and IL-15, which can be used alone or in variouscombinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, orIL-2, IL-7 and IL-15. IL-2 is available from Chiron, Emerwlle, Calif.,whereas IL-7 is available from Cytheris, Vanves, Frances. IL-15 can beobtained from PeproTech, Inc., Rocky Hill, N.J.

The T-cell growth factor can be administered by any suitable route. Ifmore than one T-cell growth factor is administered, they can beadministered simultaneously or sequentially, in any order, and by thesame route or different routes. According to one embodiment, the T-cellgrowth factor, such as IL-2, is administered intravenously as a bolusinjection. A typical dosage of IL-2 is about 720,000 IU/kg, administeredthree times daily until tolerance.

The T cell lines may be administered in conjunction withnonmyeloablative lymphodepleting chemotherapy. The nonmyeloablativelymphodepleting chemotherapy can comprise the administration ofcyclophosphamide and fludarabine, particularly if the cancer ismelanoma. A preferred route of administering cyclophosphamide andfludarabine is intravenously. Likewise, any suitable dose ofcyclophosphamide and fludarabine can be administered.

The T cell lines described herein may be stored individually or may becomprised in a bank, each cell line being categorized according to aparticular parameter (e.g. according to the HLA type of the pathogeniccell used for activation).

Thus, according to still another aspect of the present invention thereis provided a method of producing a T cell line bank comprising:generating the T cell lines described herein from a plurality ofsubjects to obtain a plurality of separate T cell lines and storing theT cell lines.

The T cell line bank of this aspect of the present invention is aphysical collection of one or more T cell lines derived from patientswith a particular disorder (e.g. cancer). Such banks preferably containmore than one sample (i.e., aliquot) of each T cell line. The bank mayalso contain one or more samples of the feeder cells, expansion agentand/or serum used to expand the MSC populations.

The T cell lines are stored under appropriate conditions (typically byfreezing) to keep them alive and functioning. According to oneembodiment, the T cell lines are stored as cryopreserved populations.Other preservation methods are described in U.S. Pat. Nos. 5,656,498,5,004,681, 5,192,553, 5,955,257, and 6,461,645.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Generation of Specific Cytotoxic T Cell Lines Against BreastCancer Cells MCF-7 and T47D Materials and Methods

Breast Cancer Cell Cultures

The MCF-7 and T47D human breast cancer cell lines were maintained inmonolayer cultures in RPMI-1640 medium supplemented with 10% fetal calfserum. For passages, confluent monolayer cultures were trypsinized withtrypsin/EDTA solution (0.25% and 0.05%, respectively), washed once, andseeded in culture medium.

Preparation of PBMC Buffy coats from blood bank donors were layered ontoLymphoprep solution (Nycomed, Oslo, Norway) and spun at 2000 rpm for 20minutes. The interface layer was collected, washed twice, counted, andresuspended in PBS; pH 7.4 to the desired cell concentration.

Breast Cancer Cells and PBMC Co-Culture In Vitro, for Primary Activation

MCF-7 and T47D were maintained in RPMI+fetal calf serum (10%). One dayprior to the start of the experiment, the medium was replaced withRPMI+human AB serum (10%). PBMC (1×10⁶) were added to MCF-7 or T47D(0.1×10⁶) at Effector/Target (E/T) 10:1 ratio, in a final volume of 1 mldispensed into 24-well microtiter plates. The cells were treated withC24D (30 μg/ml) at 0, 24 and 48 hours. The cell cultures withouttreatment were used for comparison. Culture Plates were subjected tomicroscopic evaluation on experimental days 5 and 7.

Development of a Specific Anti-Breast Cancer Cytotoxic Cell Lines (CTL)

After tumor cell cytolysis was observed in the primary culture (days5-7), the nonadherent cells (tumor and PBMC) were removed from thewells, and fresh medium containing human IL-2 (5 ng/ml) was added to thewells containing the remaining adherent cells. The cultures were treated3 times a week with RPMI-1640 containing 10% human AB serum and IL-2 (5ng/ml). The outline for generation of specific cytotoxic T cell lines ispresented in FIG. 1.

Tumor Cell Cytotoxicity and Apoptosis Assays

MTT Viability Test:

MTT reduction, which is an indicator of cellular metabolic activity, wasmeasured, as previously described (Berridge et al., Biochemica 1996;4:15-20). In brief, breast cancer cells were incubated for 4 h withlymphocytes at different E:T ratios. At 2 h incubation, 50 μl of 0.25%(w/v) solution of MTT in PBS buffer (NaCl 136.9 mM, KCl 2.68 mM, Na2HPO48.1 mM, KH2PO4 1.47 mM, pH 7.4) was added to the media and furtherincubated for 2 h. At the end of this incubation, the nonadherent cellswere removed and the remaining adherent tumor cells were washed twicewith PBS, and dissolved in a mixture of dimethyl-sulfoxide(Sigma-Aldrich), 5% (w/v) sodium dodecyl sulfate (SDS) and 1% (v/v) 1Nhydrochloric acid. After an additional brief agitation on a microtiterplate shaker, we measured the absorption at 570/650 nm with a platereader (FluoStar, BMG Labtechnology, Offenburg, Germany).

Data Analysis:

The viability was calculated with regard to the untreated breast cancercells only, control [y0], which was set to 100% viability. A lysiscontrol [y100], wherein the cells were treated with 0.5% triton X-100,was set to 0% viability. This was found to be sufficient to induce 100%cell death. Mean values from 8 wells were determined.

Annexin V Test for Apoptosis:

The ability of the specific anti-T47D CTL to induce apoptotic cell deathin T47D breast cancer cells was evaluated using Annexin V apoptosis, aspreviously described (Vermes et al., J Immunol Methods 1995; 184:39-51).In brief, 5×10⁵ tumor cells were plated in a 24-well microtiter plate.This was followed by the addition of 1.7×10⁵ CTL (E:T ratio 1:3) in 0.4RPMI medium containing 10% human AB serum. The plate was centrifuged at1300 rpm for 4 min, and the mixtures were either immediately cooled onice (5 min sample) or further cultured for 2 hours at 37° C. The cellswere collected and further reacted with FITC Annexin and propidiumiodide (PI) V/PI Detection Kit-FITC (eBioscience), according to themanufacturer's instructions.

Light-scatter characteristics were used to distinguish the tumor cellsfrom the lymphocytes, such that only the tumor cells were counted in theanalysis and the percentage of FITC-conjugated Annexin V-positive cellswas analyzed by flow cytometry (Becton Dickinson).

Results Primary Activation of Anti-MCF-7 and T47D Breast Cancer T Cellsby C24D Treatment In Vitro

T47D and MCF-7 tumor cells grew as a monolayer in tissue culture plates,as demonstrated by microscopic examination (FIGS. 2A, 3A, 4A, 5A). Theaddition of PBMC to tumor cells at E:T ratio of 10:1, did not affecttheir growth at days 5 and 7 of the culture (FIGS. 2B, 3B, 4B, 5B). Incontrast, C24D treatment of MCF-7 and T47D cells, cultured with PBMC for5-7 days at 37° C., resulted in lysis of the cancer cells as seen inmicroscopic examination (FIGS. 2C, 3C, 4C, 5C) and by a cytotoxic assaywhich showed cytolysis of 25-33% of T47D and MCF7 tumor cellsrespectively, (FIG. 6).

Following tumor cells cytolysis, immune cells remained adherent on theculture plates (FIGS. 2C, 3C, 4C, 5C). Neither cytolysis nor presence ofimmune adherent cells was observed in the untreated control cultures oftumor cells and PBMC (FIGS. 2B, 3B, 4B, 5B). The adherent cells wereidentified by FACS analysis as CD3⁺ T cells and CD14⁺ macrophages.

In Vitro Generation of Breast Cancer Cytotoxic T Cell Lines (CTL)

The adherent CD3⁺ T cells observed following tumor cells cytolysisstarted to proliferate following IL-2 addition to the 7 day primaryC24D-treated tumor-lymphocyte cultures. It is noteworthy that residualtumor cells which remained in the culture underwent complete cytolysisfollowing IL-2 addition and further T cell proliferation in the culture.The T cells multiplied exponentially in continued culture mediumcontaining IL-2 without further C24D treatment, (doubling time 48hours). The cytotoxic T cell lines were frozen and stored in liquidnitrogen for further use. In untreated tumor-PBMC cultures (control), nocytotoxic T cell lines were developed.

Anti-T47D Tumor Cell Cytotoxicity

The extent of immunity elicited by the induced anti-T47D CTL wasanalyzed by microscopic examination (FIG. 7B). It was also measured bythe MTT quantitative cytotoxic assay. After 4 hours incubation ofanti-T47D CTL with cancer cells at an E:T ratio 1:5, 60% of T47D cellswere lysed. The cytotoxic activity of the CTL was increased incomparison to that of the primary activation cultures induced at E:Tratio of 10:1, resulting in 25% T47D tumor cell cytotoxicity after 7days in culture.

It was further revealed by microscopic examination and cytotoxic teststhat the anti-T47D CTL was tumor specific and was not cytotoxic to MCF7cells (FIGS. 7D, 8).

Induction of Apoptosis of T47D Tumor Cells In Vitro

In the early stages of apoptosis, changes occur at the cell surface. Oneof the plasma membrane alterations is the translocation ofphosphatidylserine (PS) from the inner side of the plasma membrane tothe cell.

Annexin V is a Ca+ dependent phospholipid-binding protein with highaffinity for PS. Hence, this protein can be used as a sensitive probefor PS exposure on the cell membrane.

T47D cytotoxic cell line induced early apoptosis of T47D tumor cells, asevidenced by the appearance of 26.2% Annexin V positive tumor cellsafter 2 hours of incubation at E:T ratio of 1:3, as compared to 3.4% at0 h (FIGS. 9A-9B, 10).

Example 2 Broadly Reactive Cytotoxic T Cell Lines (CTL) for HLA Class IRestricted Breast Cancer Materials and Methods

Breast Cancer Cell Cultures

The MCF7, T47D and MDA-MB231 human breast carcinoma cell lines weremaintained in monolayer cultures in RPMI-1640 medium supplemented with10% fetal calf serum. For passages, confluent monolayer cultures weretrypsinized with trypsin/EDTA solution (0.25%/0.05%, respectively),washed once, and seeded in culture medium.

Anti-MCF7 and anti-T47D CTL were maintained in cultures treated 3 timesa week with RPMI-1640 containing 10% human AB serum and IL2 (5 ng/ml).

Cytokine Production Evaluation

Breast cancer cells (MCF7, T47D and MDA-MB231) were incubated for 5-20hours with CTL at different E/T ratios. At the end of the incubation,supernatants were collected and cytokine levels were measured in ELISA.

The ELISA kits for the human cytokines TNF-α and IFN-γ were purchasedfrom DPC, and R&D Systems, USA. These kits were used to quantify theindicated cytokines production in the supernatants, according to themanufacturer's instructions.

MTT Viability Test

Breast cancer cells (MCF7, T47D and MDA-MB231) were incubated for 5hours with CTL at different E/T ratios. Initially, fresh medium wasapplied to the treated cells. After 5 hours, 50 μl of a 0.25% (w/v)solution of MTT in phosphate buffered saline (PBS) buffer (NaCl 136.9mM, KCl 2.68 mM, Na2HPO8.1 mM, KH2PO4 1.47 mM, pH 7.4) was added to themedia. Two hours later, the nonadherent cells were removed and theremaining adherent tumor cells were washed twice with PBS, and dissolvedin a mixture of dimethyl-sulfoxide (Sigma-Aldrich), 5% (w/v) sodiumdodecyl sulphate (SDS) and 1% (v/v) 1N hydrochloric acid. After anadditional brief agitation on a microtiter plate shaker, the absorptionat 570/650 nm was measured with a plate reader (FluoStar, BMGLabtechnology, Offenburg, Germany).

Data Analysis

The viability was calculated with regard to the untreated breast cancercell only, control [y0], which was set to 100% viability. A lysiscontrol [y100], where the cells were treated with 0.5% triton X-100 wasset to 0% viability, which was found to be sufficient to induce 100%cell death. Mean values from eight wells were determined.

Results MDA-MB231 Cytotoxicity by Anti-MCF7 CTL

The extent of MDA-MB231 cytotoxicity by anti-MCF7 CTL followingre-stimulation, was analyzed by microscopic examination (FIGS. 11A-11D).

Cytotoxicity of MDA-MB231 cells was observed only when treated with theHLA-A2⁺ identical anti-MCF7 CTL (FIG. 11A), but not with anti-HLA-A2⁻T47D CTL (FIG. 11B). Control non-CTL PBMC were not cytolytic toMDA-MB231 cells (FIGS. 11C,D).

Cytotoxicity was also measured quantitatively by the MTT assay. As seenin FIG. 12, incubation of anti-MCF-7 CTL with both MCF7 and MDA-MB231target cells at E:T ratio 1:1 for 5 hours resulted in 70% and 80%cytotoxicity, respectively, whereas anti-T47D CTL activity wasrestricted to T47D cells. These results demonstrate that anti-MCF-7 CTLis broadly reactive, tumor specific and HLA-A2⁺ class I restricted CTL.

MDA-MB231 Restimulates MCF7 CTL to Secrete Interferon-γ

The magnitude of antigenic re-stimulation of different CTL was measuredby the level of interferon-γ secreted by anti-MCF7 and anti-T47D CTLfollowing incubation with their respective tumor target cells, as wellas with MDA-MB231. As seen in FIG. 13, re-stimulation of anti-MCF7 andanti-T47D CTL with their respective target cells resulted in increasedInterferon-γ secretion. In addition, high levels of interferon γ weremeasured following cross stimulation of anti-MCF-7 CTL with MDA-MB231.The amount of interferon secreted was proportional to the ratio of CTLeffector to tumor target (FIG. 14).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. An isolated cytotoxic T cell line which comprisesa multimeric peptide attached to an outer surface of T cells of the Tcell line, said multimeric peptide comprising at least two peptidemonomers linked to one another, each of said at least two peptidemonomers comprising at least 6 consecutive amino acids from the aminoacid sequence as set forth in SEQ ID NO: 1, wherein said at least twopeptide monomers are each no longer than 30 amino acids.
 2. A method oftreating a disease which is amenable to treatment by adoptiveimmunotherapy in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of thecytotoxic T cell line of claim 1, thereby treating the disease.
 3. Themethod of claim 2, wherein the disease is cancer.
 4. The method of claim3, wherein the cancer of the subject expresses at least one HLA class Iallele which is identical to a HLA class I allele expressed on cancercells used to generate the cytotoxic T cells.
 5. The method of claim 2,wherein the cytotoxic T cell line is generated using PBMCs.
 6. Themethod of claim 5, wherein said PBMCs are autologous to the subject. 7.The method of claim 3, wherein the cancer of the subject is selectedfrom the group consisting of breast cancer, colon cancer, lung cancer,Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphatic leukemia(ALL) and renal cancer.
 8. The method of claim 7, wherein said breastcancer comprises triple negative breast cancer.
 9. The method of claim2, wherein said cytotoxic T cell line is generated by incubating T cellswith pathogenic cells in the presence of a multimeric peptide comprisingat least two peptide monomers linked to one another, each of said atleast two peptide monomers comprising at least 6 consecutive amino acidsfrom the amino acid sequence as set forth in SEQ ID NO: 1, wherein saidat least two peptide monomers are each no longer than 30 amino acids,wherein the multimeric peptide is capable of reducing binding of PLIF tohuman leukocytes under conditions which allow expansion of said T cells.10. A bank comprising a plurality of the cytotoxic T cell lines of claim1.